Identification and Safety Assessment of Enterococcus casseliflavus KB1733 Isolated from Traditional Japanese Pickle Based on Whole-Genome Sequencing Analysis and Preclinical Toxicity Studies.

The present study involves the precise identification and safety evaluation of Enterococcus casseliflavus KB1733, previously identified using 16S rRNA analysis, through whole-genome sequencing, phenotypic analysis, and preclinical toxicity studies. Analyses based on the genome sequencing data confirm the identity of KB1733 as E. casseliflavus and show that the genes related to vancomycin resistance are only present on the chromosome, while no virulence factor genes are present on the chromosome or plasmid. Phenotypic analyses of antibiotic resistance and hemolytic activity also indicated no safety concerns. A bacterial reverse mutation test showed there was no increase in revertant colonies of heat-killed KB1733. An acute toxicity test employing heat-killed KB1733 at a dose of 2000 mg/kg body weight in rats resulted in no deaths and no weight gain or other abnormalities in the general condition of the animals, with renal depression foci and renal cysts only occurring at the same frequency as in the control. Taking the background data into consideration, the effects on the kidneys observed in the current study were not caused by KB1733. Our findings suggest that KB1733 is non-pathogenic to humans/animals, although further studies involving repeated oral toxicity tests and/or clinical tests are required.

Safety evaluation of arabinase (arabinan endo-1,5-α-L-arabinanase) from Aspergillus tubingensis.

Arabinase is an enzyme recognized for its ability to degrade arabinan, a plant cell wall constituent. It has been applied in the food industry most commonly for juice processing. One commercial source of arabinase is Aspergillus tubingensis (A. tubingensis), a black Aspergillus species. Given the intended use in food for human consumption, and noting its potential presence at trace levels in finished products, a series of safety studies including in vitro Ames and chromosome aberration assays, in vivo mammalian erythrocyte micronucleus and alkaline comet assays, and a 90-day rat oral toxicity study were conducted. No test article-related mutagenic activity was observed in the Ames assay. Although positive activity was observed in the chromosome aberration assay, this was not replicated in the in vivo genotoxicity assays including in preabsorptive cells. In the subchronic toxicity study, no test article-related adverse effects were observed following oral administration of arabinase at doses of 15.3, 153, or 1,530 mg total organic solids (TOS)/kg body weight/day to Sprague Dawley rats. The no-observed-adverse-effect level was considered to be the highest dose tested (1,530 mg TOS/kg body weight/day). The results of the genotoxicity studies and the subchronic toxicity study support the safe use of arabinase from A. tubingensis in food production.

Safety evaluation of AMP deaminase from Aspergillus oryzae.

Adenosine-5'-monophosphate (AMP) deaminase is an enzyme used to increase concentrations of 5'-inosine monophosphate in certain foods and beverages for flavoring purposes. One commercial source of this enzyme is Aspergillus oryzae, a filamentous fungus with a history of safe use in Asia as a fermentation organism used in the production of miso sauce and sake liquors. Noting the use of the enzyme in food intended for human consumption and potential presence at trace levels in finished goods, a series of safety studies including an in vitro Ames test and chromosome aberration assay with Chinese hamster lung fibroblasts were conducted along with a 90-day oral toxicity study in rats. AMP deaminase showed no evidence of genotoxicity in the in vitro tests. Following gavage administration of Sprague-Dawley rats at dosages of 19.8, 198.4, or 1984 mg total organic solids (TOS)/kg body weight (bw)/day for 90 days, no adverse effects on body weight gain, food consumption, hematology, clinical chemistry, urinalysis, ophthalmological and histopathological examinations were observed. The no-observed-adverse-effect level was considered to be 1984 mg TOS/kg bw/day, the highest dose tested. Results of the genotoxicity studies and subchronic rat study support the safe use of AMP deaminase produced from A. oryzae in food production.